The green fluorescent protein (GFP) is a 238 amino acid molecule which is the ultimate source of fluorescent light emission in the jellyfish Aequorea victoria. The GFP excitation spectrum shows an absorption band (blue light) maximally at 395 nm with a minor peak at 470 nm, and an emission peak (green light) at 509 nm. The longer-wavelength excitation peak has greater photostability then the shorter peak, but is relatively low in amplitude; Chalfie et al., Science, 263:802-805 (1994). The GFP absorption bands and emission peak arise from an internal p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of a Ser-Tyr-Gly sequence at residues 65-67; Cody et al. Biochemistry 32:1212-1218 (1993).
The gene for GFP was first cloned by Prasher et al., Gene, 111:229-233 (1992), and cDNA for the protein produces a fluorescent product identical to that of native protein when expressed in prokaryotic (E. coli) and eucaryotic (C. elegans) cells; Chalfie et al., Science, 263:802-805 (1994). Importantly, exogenous substrates and cofactors are not required for GFP fluorescence in such cells; Id. As such, GFP is considered to have tremendous potential in methods to monitor gene expression, cell development, or as an in situ tag for fusion proteins; Heim et al., P.N.A.S. USA, 91:12501-12504 (1994).
Chalfie and Prasher, WO 95/07463 (March 16, 1995), describe various uses of GFP, including a method of examining gene expression and protein localization in living cells. Specifically, methods are described wherein: 1) a DNA molecule is introduced into a cell, said DNA molecule having DNA sequence of a particular gene linked to DNA sequence encoding GFP such that the regulatory element of the gene will control expression of GFP; 2) the cell is cultured in conditions permitting the expression of the fused protein; and 3) detection of expression of GFP in the cell, thereby indicating the expression of the gene in the cell. Methods such as those described by Chalfie and Prasher are advantageous compared to previously reported methods which utilized .beta.-galactosidase fusion proteins; see e.g. Silhavy and Beckwith, Microbiol. Rev., 49:398 (1985); Gould and Subramani, Anal. Biochem., 175:5 (1988); Stewart and Williams, J. Gen. Microbiol., 138:1289 (1992), or luciferases; Id., in that the need to fix cell preparations and/or add exogenous substrates and cofactors is eliminated.
Several groups have studied various GFP mutants in order to identify a GFP having improved fluorescent properties. For example, Heim et al., P.N.A.S USA, 91:12501-12504 (1994) report on GFP variants having significant alterations in the ratio of the two main wildtype excitation peaks. In particular, a Ile.sup.167 .fwdarw.Thr mutant had increased fluorescence at 475 nm excitation. Also identified was a mutant, Tyr.sup.66 .fwdarw.His, which fluoresced blue.
Heim et al., Nature, 373:663-664 (1995) report that simple point mutations in Aequorea GFP bring its spectra closer to that of Renilla reniformis GFP, a protein with only one absorbance and excitation peak. In particular, a Ser.sup.65 .fwdarw.Thr mutant showed greatly increased brightness and rate of fluorophore generation as compared to wildtype Aequorea GFP.
Delagrave et al., BIO/TECHNOLOGY, 13:151-154 (1995) report on several Aequorea GFP variants that showed red-shifted excitation spectra similar to that of Renilla reniformis GFP, i.e., shift in excitation maxima from 393 nm to 498 nm. Delagrave et al. hypothesize that co-expression of GFP and red-shifted GFP (RSGFP) will enable the analysis of two proteins or promoters per cell or organism.
To date, there have been no reports of temperature sensitive GFP mutants having enhanced fluorescence at high temperatures, e.g., 37.degree. C., where wildtype GFP does not fluoresce well. Such mutants would provide obvious and significant advantages for use as cell markers or protein expression indicators in prokaryotic and, especially, eucaryotic systems where the standard physiological temperature is 37.degree. C.